HGF激活mTOR信号通路促进ARDS肺微血管内皮修复的机制研究

The function of pulmonary microvascular endothelial cell (PMVEC) barrier stabilized is a therapeutic approach to improve the injury in ARDS, as acute respiratory distress syndrome (ARDS) is characterised by endothelial hyperpermeability. Our previous studies demonstrated that mesenchymal stem cells (MSC) can significantly improve the permeability and increase the proliferation of PMVEC through their paracrine effects of hepatocyte growth factor (HGF), but the specific mechanism is still uncertain. Previous studies have showed that HGF could lead to the phosphorylation of mTOR by combining its membrane receptor c-Met, promoting the repairment of endothelial cell injury and improving cell proliferation. PMVEC have been proved containing c-Met in our pilot study. However, there is still a lack of correlation between mTOR pathway and PMVEC repair in ARDS. Therefore, we hypothesized that HGF activating mTOR signaling pathway is an important mechanism for improving the repair of PMVEC in ARDS. The purpose of this study are as below: ①To determine that it is an important mechanism of mTOR signaling pathway activation by HGF in the repairment of ARDS-PMVEC in vitro by interfering the expression of mTOR and its subtypes in PMVEC by lentiviral vector transfection and siRNA techniques; ②To demonstrate the different roles of mTORC1 and mTORC2 in the repairment of ARDS-PMVEC in vitro; ③To prove the significant mechanism of mTOR signaling pathway activated by HGF in improving ARDS in vivo using mTOR knockdown ARDS mice model in order to provide new theoretical basis for ARDS in clinical practice.

肺微血管内皮（PMVEC）通透性增加是ARDS主要病理改变，促进其修复是改善ARDS重要手段。我们前期研究表明，MSC旁分泌HGF可显著改善PMVEC通透性并增加其增殖活力，但机制不明。既往研究显示HGF可与其受体c-Met相结合，介导mTOR磷酸化，促进内皮细胞修复、调控细胞增殖。我们已经证实PMVEC中含有c-Met，但mTOR通路在ARDS-PMVEC修复中的作用缺乏研究。本研究假设：HGF激活mTOR信号通路是促进ARDS-PMVEC损伤修复的重要机制。研究拟①采用慢病毒转染及siRNA技术调控PMVEC上mTOR及其亚型表达，体外证实mTOR信号通路修复肺血管内皮细胞的重要机制；②探讨mTOR不同亚型对修复ARDS-PMVEC的不同作用；③构建mTOR敲除的ARDS小鼠模型，在体证实mTOR信号通路在HGF促进ARDS小鼠PMVEC修复的重要作用，为临床治疗提供理论依据。

肺微血管内皮损伤是ARDS主要的病理改变，促进其修复是改善ARDS重要手段。课题组发现MSC旁分泌HGF可显著改善ARDS肺微血管内皮细胞通透性，但具体机制仍不清楚。既往研究表明HGF结合c-Met，能够介导mTOR磷酸化，调控细胞增殖。我们将重组HGF与LPS刺激的内皮细胞共培养，发现HGF能够促进mTOR蛋白磷酸化，缓解内皮细胞凋亡。雷帕霉素抑制mTOR蛋白活化，阻断HGF的修复作用。线粒体损伤是LPS引起细胞凋亡的重要机制，HGF与损伤的内皮细胞共培养，能够显著降低钙离子内流，减少ROS产生，缓解内皮细胞凋亡和细胞焦亡。而抑制mTOR活化或STAT3活化，均能够阻断HGF的修复作用。mTOR蛋白作为功能性亚基，在细胞内主要参与两种分子复合物的形成，即mTORC1和mTORC2，发挥相应的功能。然而，mTORC1和mTORC2分别在HGF促进ARDS肺血管内皮损伤修复中的作用仍不清楚。为此，课题组通过慢病毒感染内皮细胞干扰Raptor、Rictor表达，分别阻断mTORC1和mTORC2形成，进一步增强LPS导致的细胞旁通透性增强和跨内皮细胞通透性增强，阻断HGF的修复作用。动物实验表明，尾静脉注射重组HGF能够显著缓解脓毒症小鼠肺内炎症，缓解急性肺损伤，降低小鼠病死率。研究提示，HGF通过活化mTOR信号通路，促进肺微血管内皮细胞损伤修复，改善ARDS肺组织损伤，有可能是未来ARDS治疗的重要策略。为进一步明确血浆中HGF水平与脓毒症患者病情及预后的相关性，检测86例脓毒症患者的血浆HGF水平，发现脓毒症28天内死亡患者血浆HGF水平显著高于存活者，而HGF水平与内皮损伤相关标志物vWF、肺血管通透性指数PVPI、以及液体入量、液体平衡和SOFA评分正相关，与体循环阻力指数SVRI负相关，提示血浆HGF水平可能是反映脓毒症血管内皮损伤的重要标志物。此外HGF水平预测脓毒症患者28天预后的曲线下面积达0.722，阈值为1832pg/mL，血浆HGF水平升高可能提示患者预后死亡风险增加。